2,3–Dihydroxybenzoic Acid Decarboxylase from Aspergillus niger

Abstract

2,3‐Dihydroxybenzoic acid decarboxylase, the last enzyme in the fungal metabolism of indole to catechol, catalyzes the non‐oxidative decarboxylation of 2,3‐dihydroxybenzoic acid to catechol. Unlike most other decarboxylases, this enzyme does not require a cofactor, underlining the importance of active‐site residues in the reaction mechanism. Earlier studies from this laboratory [Kamath, A. V., Appaji Rao, N. & Vaidyanathan, C. S. (1989) Biochem. Biophys. Res. Commun. 165, 20–26], have shown that the sulfhydryl agent N‐ethylmaleimide (MalN Et) inactivated the enzyme by modifying a single class of cysteine residues and that this inactivation was prevented in the presence of salicylate, a substrate analogue. In the present study, this essential cysteine residue has been identified by specific labelling with [14C]‐MalN Et using the differential labelling technique. The stoichiometry of incorporation of [14C]MalN Et was approximately one/subunit of the homotetrameric protein. The peptide bearing this reactive cysteine residue was isolated by tryptic digestion of the differentially labelled enzyme and subsequent reverse‐phase chromatography of the peptide mixture. The sequence of the major radioactive peptide that was identified to be the active‐site peptide, was LLGLAETCK. A search for sequences similar to this active‐site peptide indicated that this sequence was probably unique to the decarboxylase under study. A partial primary structure map constructed from the sequences of peptides derived from enzymic cleavage of the protein using endoproteinase Glu‐C and trypsin did not share any significant sequence similarity with sequences reported in the database, again suggesting the uniqueness of the enzyme. This is the first report on the active‐site peptide and the partial primary structure of a non‐oxidative decarboxylase catalyzing the removal of a carboxyl group from an aromatic nucleus.